Pseudomonas aeruginosa is a gram-negative human pathogen that frequently causes severe hospital-acquired infections. Despite appropriate antibiotic therapy, Pseudomonas infections are associated with high rates of mortality. Pseudomonas is observed to invade epithelial cells in vitro and in vivo. Although the clinical significance of this observation is not understood, Pseudomonas internalization has been postulated to play a role in such diverse processes as local host defense mechanisms versus bacterial evasion of antibiotics and the immune system. The objective of this project is to describe the mechanism by which Pseudomonas invades epithelial cells: a first step toward our long-term objective of understanding the role bacterial invasion plays in clinical disease. This proposal describes two complementary series of experiments which will define both the bacterial and eukaryotic components required for Pseudomonas invasion of epithelial cells in vitro. In Specific Aim number 1, a genetic screen will be used to identify bacterial gene products required for invasion; these molecules, which will include novel Pseudomonas adhesins and invasins, will be characterized and then used to isolate their eukaryotic receptors. A separate series of experiments described in Specific Aim number 2 will identify the eukaryotic pathway responsible for actin-dependent uptake of Pseudomonas. Epithelial cells expressing constitutively active and dominant negative alleles of the small GTPases rac1, rhoA and cdc42 and of dynamin will be used to examine the roles of actin cytoskeleton rearrangements and receptor-mediated endocytosis in Pseudomonas internalization. The results of the proposed experiments are likely to have an impact on our ability to understand and treat P. aeruginosa infections. The bacterial adhesins and invasins identified by this approach will be excellent targets for novel anti-pseudomonal therapies; these are much needed given the increasing antibiotic resistance exhibited by clinical Pseudomonas isolates. The tropism of Pseudomonas for injured epithelial tissues may be better understood once the bacterial ligands and eukaryotic receptors governing Pseudomonas-host cell interactions are known; these may also be developed into targets for therapies designed to prevent Pseudomonas colonization and disease in individuals at high risk, such as burn patients.